Analysis of ET-A and ET-B receptors using an isolated perfused rat lung preparation.

AIMS AND METHODS: The pulmonary and vascular effects of endothelin-1 receptor activation were studied in isolated perfused and ventilated lung preparations from rat. The responses to endothelin-1 (ET-1) and the endothelin B (ET(B)) receptor agonist sarafotoxin 6c (S6c) were characterized using the endothelin A (ET(A))-receptor antagonist FR 139317, the ET(B)-receptor antagonist BQ 788 and the combined ET(A)/ET(B)-receptor antagonist Bosentan. The respiratory parameter airway conductance (G(aw)) and the vascular parameter perfusion flow were analysed simultaneously. RESULTS: Concentration-response curves for ET-1 administered intra-arterially revealed that its most potent effect was on the vascular side while S6c had a more potent effect on airway conductance. ET-1, given as a bolus dose intra-arterially (100 microL of 0.2 nM), induced a strong- and long-lasting contraction of the vasculature while only a less pronounced contraction was seen in the airways. Neither of the antagonists had a significant effect per se on G(aw) or perfusion flow. FR 139317 reduced the effect of ET-1 on perfusion flow by about 50%, while airway conductance was augmented. BQ 788 enhanced the decrease in perfusion flow by ET-1 while G(aw) was not influenced. The combined ET(A)/ET(B) antagonist Bosentan powerfully prevented the ET-1-induced decrease in G(aw) but did not alter its reduction in perfusion flow. CONCLUSIONS: The potent effect of ET-1 on the vascular side of the lung is mediated mainly through ET(A) receptors, whereas both ET(A) and ET(B) receptors are involved in G(aw) in the rat lung.

The role of P-glycoprotein in limiting intestinal regional absorption of digoxin in rats.

The objective of this work was to study the role of regional intestinal efflux activity of P-glycoprotein (Pgp) in situ in anesthetized rats in limiting the absorption of digoxin. A 10-cm portion of duodenum or jejunum, or 5-cm of colon was perfused single-pass with saline containing [(3)H]digoxin while the appearance of radioactivity in the blood was measured. Verapamil in the perfusate was used as a modulator of Pgp in the intestinal mucosa. Net water absorption, mucosal integrity, and intestinal motility of the isolated segment were monitored, as well as heart rate and blood pressure. Excretion of i.v. administered unlabelled digoxin, 1 mg/kg, into the intestine while perfusing the duodenum-proximal jejunum region, was studied for comparison. At a perfusate concentration of 1 mM, verapamil caused a dramatic increase in [(3)H]digoxin absorption rate from duodenum and jejunum, while the effect in colon was insignificant. At concentrations of 0.1, 1, and 2.5 mM in the duodenal perfusate, verapamil increased the absorption rate of [(3)H]digoxin in a dose-dependent manner. The lowest concentration almost doubled the rate without having any significant effects on the cardiovascular system, intestinal motility, or net absorption of water. The excretion rate of unlabelled digoxin from the blood into the gut lumen was found to be halved in the presence of 0.5 mM verapamil in the perfusate. Absorption rate of [(3)H]digoxin in the rat is likely limited by Pgp-mediated efflux. The data indicate that Pgp plays an important role for digoxin efflux in the small intestine only.

Leukocyte sequestration in isolated guinea pig lungs during extracorporeal circulation: effects on microvascular function.

Neutrophils accumulate in patient lungs during clinical hemodialysis and in isolated blood-perfused guinea pig lungs due to the contact between blood and extracorporeal system. However, it is unclear how these sequestered and partly activated neutrophils affect the lung microvasculature. We, therefore, studied pulmonary vascular resistance, vascular permeability, gas exchange, and oxygen free radical production in isolated guinea pig lungs during perfusion with whole blood containing partly 'activated' neutrophils in comparison with perfusions using leukopenic blood. We also connected a Cuprophan hemodialysis membrane to the whole-blood perfusion system in order to investigate whether a dialyzer, which may further activate leukocytes, affects lung microvascular permeability, vascular resistances, and reactive oxygen species production. The sequestered neutrophils did not seem to markedly affect the lung microvascular function, since neither the leukocyte-free perfusion nor the hemodialysis membrane altered any of the measured variables as compared with whole-blood perfusion in a system without a dialyzer. We conclude that neutrophils, whether activated by a perfusion system or by a dialysis membrane, can accumulate in isolated lungs without adversely affecting the microvascular function.

An isolated blood-perfused guinea-pig lung model for simultaneous registration of haemodynamic, microvascular and respiratory variables.

We have developed an optimized isolated lung perfusion system, which possesses several advantages. Firstly, studies of microvascular, respiratory, haematological and biochemical variables are combined in one model. Secondly, blood perfusion resulted in less oedema formation than buffer-perfused lungs, and high PO2 through ventilation with room air. Finally, data for the variables can be displayed, controlled and recorded in real time using a computerized system permitting subsequent processing (e.g. filtering without destroying original data). In this paper we discuss the basic behaviour of the model in terms of vascular resistance, vascular permeability, respiration and neutrophil sequestration. In addition, the effects of oleic acid, histamine and histamine receptor blockers were tested, and two methods of calculating vascular permeability are discussed. The way in which different anaesthetics affect the neutrophil content of lung tissue and blood was also investigated. In the model, oleic acid increased pulmonary vascular resistance and permeability, whereas histamine did not affect either permeability or the pre/postcapillary vascular resistance ratio. However, histamine receptor blockers increased this ratio, indicating that there was endogenous histamine release. The neutrophil content of the isolated lungs was increased, but this did not affect the variables measured. There was also accumulation of neutrophils in the lungs of blood donor animals, due to CO2 sedation. However, CO2 sedation proved to be superior to pentobarbital or ketamine anaesthesia in maintaining the levels of neutrophils circulating in the blood. In conclusion, this model seems to be sensitive and to yield reproducible results regarding the physiology or pathophysiology of the lung.

Platelet activating factor-induced pulmonary accumulation of 111Indium-oxine labelled neutrophils in anesthetized guinea pigs.

The thoracic accumulation of neutrophils labelled with 111Indium-oxine in response to infusion of platelet activating factor (PAF, 18 ng/kg/min x 5 min, i.v.) was studied using an automated isotope monitoring system (AIMSplus) in anesthetized guinea-pigs. Loss of cell associated radioactivity in vitro was less than 1% over 4 hr. Labelled neutrophils maintained their functional capacity (oxidative response to the cell stimulants N-formyl-L-methionine-L-leucine-L-phenylalanine and phorbol myristate acetate) and greater than 95% viability (ethidium bromide/acridine orange stain) in vitro. Total thoracic radioactivity increased significantly from baseline in response to PAF with a slight tachyphylaxia in the neutrophil-accumulation after a repeat PAF infusion. The highest ratios of radiolabel (tissue/blood) were found in the spleen much greater than liver greater than lung.

Intrathoracic accumulation of 111In-labeled neutrophils in guinea pigs in response to PAF.

The intrathoracic content of neutrophils, labeled with 111In-oxine has been measured in the anesthetized guinea pig by using an automated isotope-monitoring system. Intravenous infusion of platelet-activating factor (PAF; 5.6, 10, or 18 ng.kg-1.min-1 over 5 min) caused a dose-related abrupt intrathoracic accumulation of neutrophils, which dispersed from the thorax within 20 min. Repetition of this procedure after 1 h gave responses of comparable magnitude and duration. Anti-platelet antiserum pretreatment did not influence the response of neutrophils to PAF. Iloprost infusion (10 ng.kg-1.min-1 over 15 min) did not affect the response of neutrophils to PAF, whereas accumulation of radiolabeled platelets in the lung was totally suppressed by this dose. Intrathoracic accumulation of neutrophils in response to PAF can be considered to be independent of platelet activation.

Oleic acid-induced injuries in the guinea-pig. Effects of allopurinol on cell dynamics, erythrocyte-catalase and uric acid plasma levels.

1. Oleic acid was used to produce adult respiratory distress syndrome-like pulmonary microvascular injuries. The resulting injuries have previously indicated involvement of accumulating neutrophils (Hultkvist et al. 1988). Activated neutrophils release oxygen free radicals that may be possible to detect in the plasma. 2. The dynamics of neutrophils and platelets were studied in the guinea-pig after oleic acid-induced injury (0.03 ml/kg per 10 min). 3. As an indication of oxygen free radical activity, plasma levels of uric acid and red blood cell (RBC)-catalase, were analysed. 4. Allopurinol (10 mg/kg, i.p.) was given prior to oleic acid infusion to block the production of uric acid. 5. The neutropenia, in contrast to the thrombocytopenia seen at 15 min, was significantly inhibited in the allopurinol pretreated group compared with oleic acid and vehicle alone. 6. The blood plasma concentration of uric acid was significantly elevated after 15 min from start of experiment. Allopurinol pretreatment significantly reduced the uric acid plasma level. 7. The RBC catalase activity did not change with time within or between any groups. 8. The results indicate that sequestration of activated neutrophils in the microvasculature are to some extent oxygen free radical dependent.

External detection of pulmonary accumulation of indium-113m labelled transferrin in the guinea pig.

Accumulation of radioisotope labelled transferrin in the lungs of guinea pigs was determined with an external detection system. The method is based on the intravascular and extravascular distribution of indium-113m labelled transferrin compared with the intravascular distribution of technetium-99m labelled red blood cells. Guinea pigs were given iloprost, a prostacyclin analogue and potent pulmonary vasodilator, and noradrenaline, a pulmonary vasoconstrictor, in an attempt to increase and decrease respectively the blood volume in the lungs. Neither agent altered transferrin accumulation in the lung by comparison with a saline infusion. Iloprost infused before and after oleic acid infusion reduced macro-molecular leakage when compared with oleic acid alone. These data suggest that the double isotope method can distinguish between hydrostatic and injury induced pulmonary oedema.